Long non-coding RNAs involved in Drosophila development and regeneration

Abstract The discovery of functional long non-coding RNAs (lncRNAs) changed their initial concept as transcriptional noise. LncRNAs have been identified as regulators of multiple biological processes, including chromatin structure, gene expression, splicing, mRNA degradation, and translation. However, functional studies of lncRNAs are hindered by the usual lack of phenotypes upon deletion or inhibition. Here, we used Drosophila imaginal discs as a model system to identify lncRNAs involved in development and regeneration. We examined a subset of lncRNAs expressed in the wing, leg, and eye disc development. Additionally, we analyzed transcriptomic data from regenerating wing discs to profile the expression pattern of lncRNAs during tissue repair. We focused on the lncRNA CR40469, which is upregulated during regeneration. We generated CR40469 mutant flies that developed normally but showed impaired wing regeneration upon cell death induction. The ability of these mutants to regenerate was restored by the ectopic expression of CR40469. Furthermore, we found that the lncRNA CR34335 has a high degree of sequence similarity with CR40469 and can partially compensate for its function during regeneration in the absence of CR40469. Our findings point to a potential role of the lncRNA CR40469 in trans during the response to damage in the wing imaginal disc.


Introduction
Long non-coding RNAs (lncRNAs) are defined as transcripts longer than 200 nucleotides that lack protein-coding potential.They have been mostly identified in high-throughput transcriptomic studies and are quite abundant in metazoa (1)(2)(3).They tend to show low sequence conservation ( 1 ,3 ), although they may show other conservation signatures such as positional conservation or synteny, structural conservation, or functional convergence ( 4 ).Many do not produce observable phenotypes upon mutation ( 5 ,6 ), and the vast majority have not been assigned to a putative function ( 7 ).Moreover, the expression of lncRNAs is generally lower and highly tissueand stage-specific when compared to that of protein-coding genes ( 1 , 3 , 8 ), hampering their proper characterization.In recent years, a small proportion of annotated lncRNAs have been functionally characterized and described to participate in the regulation of almost every step of gene expression ( 9 ).Multiple lncRNAs are known to regulate the expression of overlapping or nearby protein-coding genes ( 10 ,11 ), but some are also capable of influencing the expression of genomically distant genes in trans ( 12 ,13 ).LncRNAs can interact with DNA, RNAs, proteins, and even membrane lipids, showing diverse subcellular distributions ( 14 ,15 ).
LncRNAs are involved in multiple developmental processes, including the regulation of Hox genes ( 16 ), the regulation of chromatin accessibility in dosage compensation mechanisms ( 17 ), and the development of organs such as the brain ( 18 ) and heart ( 19 ).Alterations in the expression of lncRNAs have also been described for several diseases, including cancer ( 20 ,21 ), cardiovascular diseases ( 22 ) and neurological disorders ( 23 ).In fact, the public database LncRNADisease v2.0 currently describes more than 1700 experimentally validated lncRNA-disease associations ( 24 ), highlighting lncRNAs as potential tools to understand the mechanisms and prognosis of multiple diseases.Moreover, changes in lncRNA expression have been found in the hypoxia response pathway and in response to various stress conditions including oxidative stress, heat-shock and DNA damage ( 25 ).
The number of annotated lncRNAs varies greatly from one species to another.While the numbers of annotated proteincoding genes and lncRNAs are similar in humans and mice, the number of lncRNAs is considerably lower in Drosophila melanogaster and Caenorhabditis elegans ( 26 ).Despite this, some lncRNA features are shared between mammals and Drosophila , including the transcript length and the proportion of genic / intergenic lncRNAs ( 26 ).Moreover, some lncRNAs function similarly in different species, such as the lncRNAs Xist and roX1 / roX2 , which recruit the chromatin-modifying complexes that drive the dosage compensation mechanisms in mammals and flies, respectively ( 17 ,27 ).
The expression of lncRNAs has been extensively characterized throughout Drosophila embryogenesis and larval development, revealing a highly temporally restricted expression profile, especially at the late embryonic and late larval stages, which are critical times for developmental transitions ( 28 ,29 ).The subcellular localization of 103 lncRNAs has also been determined over the course of embryogenesis and in the late third instar larval tissues ( 14 ).However, there is no detailed information regarding lncRNA expression in imaginal discs at different stages of development or after damage.
Drosophila imaginal discs are larval epithelial sacs that give rise to the adult structures after differentiation during metamorphosis.Each imaginal disc develops from a cluster of a few cells in the embryo and its morphology and size matures during the larval stages.Mature discs undergo major morphogenetic events during metamorphosis ( 30 ).Third instar larval imaginal discs also present a high capacity to regenerate following a physical injury ( 31 ,32 ) or after genetic ablation (33)(34)(35).Upon damage, early signals that include calcium waves and reactive oxygen species (ROS) are propagated from the dying cells to the neighboring living cells, activating a series of signaling pathways that are required for wound healing and regenerative growth (36)(37)(38).The activation of these pathways leads to a burst of active transcription at the early stages of regeneration ( 39 ), activating, among others, the transcription factor Ets21C , which is described to orchestrate a regeneration-specific gene regulatory network ( 40 ).
In this work, we analyzed the expression of lncRNAs in different imaginal discs during larval and pupal stages, as well as in the regeneration of wing discs.We found that the intergenic lncRNA CR40469 is required for regeneration, but not in development.The recovery of the regeneration capacity of CR40469 mutants with the addition of ectopic CR40469 led us to hypothesize about a putative trans -acting role after damage.Additionally, we found that CR40469 and a second lncRNA, CR34335 , share a high sequence similarity.Despite showing opposite expression profiles, the ectopic expression of CR34335 also rescued the regeneration capacity of CR40469 mutants, suggesting that it may compensate for the function of CR40469 in its absence.
A Catalan version of the abstract of the article can be found at: https:// doi.org/ 10.5281/ zenodo.13258939 .

Reagent name Source Identifier
Anti-Digoxigenin-  Genes and transcripts were quantified in transcripts per kilobase million (TPMs) using RSEM v1.2.21 ( 46 ).The average TPMs for each gene in each sample was calculated as the average TPMs for two biological replicates.Only genes with an expression of at least 1 TPM were considered expressed.Genes aligned to non-canonical chromosomes were discarded.A total of 13957 protein-coding genes and 2455 lncRNAs were considered (FlyBase genome annotation version r6.29 ( 50 ,51 )).
Genes were considered tissue-specific if their expression was at least 1 TPM in only one particular tissue, independent of the developmental stages in which they were expressed.Stage-specific genes had an expression of at least 1 TPM in only one particular developmental stage, independent of the tissues in which they were expressed.
LncRNA classification and association with protein-coding genes (PCGs) LncRNAs were classified with respect to their genome location using the classification module of the FEELnc pipeline ( 43 ).FEELnc received the 2455 annotated lncRNAs from the FlyBase genome annotation version r6.29 as input, classifying the lncRNAs into three broad groups: lncRNAs not overlapping with any other PCGs were considered intergenic, lncR-NAs located within an intron of a PCG were classified as genic intronic, and lncRNAs overlapping by at least 1 bp with an exonic sequence of a PCG were classified as genic exonic.The classification was mutually exclusive in the following rank: genic exonic > genic intronic > intergenic.
Intergenic lncRNAs were associated with the closest PCG by measuring the end-to-end distance, independent of the expression, orientation, and direction of transcription.Genic intronic and genic exonic lncRNAs were paired with their overlapping PCGs.For lncRNAs overlapping with multiple PCGs, we only considered the gene showing more overlapping nucleotides.

Expression profile and lncRNA-PCG pair classification
The expressed lncRNAs shown in Figure 1 B and their associated PCGs ( Supplemental Table S1 ) were each classified as increasing (lowest expression in L3 and highest expression in LP), decreasing (lowest expression in LP and highest expression in L3), peak (highest expression in EP) or valley (lowest expression in EP).The differentially expressed lncR-NAs in regeneration shown in Figure 3 B and their associated PCGs ( Supplemental Table S3 ) were each classified according to their expression during regeneration as increasing (lowest expression at the early stage and highest expression at the late stage), decreasing (lowest expression at the late stage and highest expression at the early stage), peak (highest expression at the mid stage) or valley (lowest expression at the mid stage).
LncRNA-PCG pairs were then classified as concordant (if the classification of both members in the pair matched: increasing / increasing, decreasing / decreasing, peak / peak and valley / valley), as discordant (if the classification of both members in the pair was opposite: increasing / decreasing and peak / valley), or as unrelated (if the PCG was not expressed or if they were not classified as concordant or discordant: increasing / peak, increasing / valley, decreasing / peak and decreasing / valley).
We used R to calculate the pairwise Pearson correlation coefficient (PCC) for each lncRNA-PCG pair.Normalized expression data (log 10 -transformed TPMs plus a pseudocount of 0.1) of the wing disc (Figure 2

Gene ontology (GO) analysis
The GO enrichment analysis tool ( 45 ) from the PANTHER 17.0 database was used to determine the GO term enrichment, using biological process trees.Input gene sets were the PCGs from concordant lncRNA-PCG pairs.P -values were adjusted using the false discovery rate correction for multiple comparisons.

Differential expression analysis in regenerating discs
Differential gene expression analyses of the control and regeneration samples, obtained from Vizcaya-Molina and colleagues ( 39 ) (GEO Accession number: GSE102841), were performed separately at each time-point.We removed all the genes expressed < 1 TPM in all samples.We used a simple fold change approach of ≥|1.7| and a DESeq2 ( 42 ) analysis considering an absolute fold change of ≥1.7 and a Benjamini-Hochberg adjusted P -value < 0.05.Genes DE using both methods were considered.Protein-coding genes rpr and Gadd45 were used as positive controls.
We obtained an initial list of 201 DE lncRNAs.The absence of strand information in these RNA-seq samples hampered the correct attribution of reads between the exonic lncRNAs and their overlapping PCGs.For this reason, we manually validated the initial 109 DE exonic lncRNAs using the RNA-seq bigwig tracks of the UCSC Genome Browser.After manual curation, we removed 70 exonic lncRNAs, ending up with a robust list of 131 DE lncRNAs ( Supplemental Table S2 ).Among them, we found 5 stable intronic sequence RNAs (sisRNAs) and 3 hairpin RNAs (hpRNAs), which were not considered for the selection of our candidate lncRNA.
Due to the 99.1% similarity between the CR40469 and CR34335 sequences, we visually inspected the reads mapped to the CR40469 locus to confirm that mapping was correct.We corroborated that the two mismatched nucleotides between both lncRNAs were sufficient to correctly map the reads to each locus.

Developmental expression profile of PCGs and lncRNAs
The expression profile of PCGs (Figure 1 E), lncRNAs (Figure 1 F) and differentially expressed lncRNAs (Figure 3 H) in developing wing, leg and eye discs was represented as a heatmap.Gene expression values were standardized into Z -score values and normalized into a -3 to 3 scale.Gene and sample clustering was performed using a complete hierarchical clustering.Major gene clusters were highlighted for each heatmap.

Generation of CR40469 knock-out mutants and UAS transgenic flies
The deletion of CR40469 was performed using the ends-out homologous recombination technique.Long homology arms for the upstream and downstream regions of the CR40469 locus were designed.A 1089-bp containing the entire CR40469 gene, as well as the upstream 663 bp and downstream 212 bp was excised and replaced by an mCherry cassette.The absence of the CR40469 locus was confirmed by PCR.The designed primer sequences are shown in Supplemental Table S5 .
For the ectopic activation of CR40469 and CR34335 , we used a semi-directed cloning protocol to clone the entire CR40469 or CR34335 genomic sequences into a pUAST plasmid.We designed primers targeting the CR40469 and CR34335 genes ( Supplemental Table S5 ).An EcoRI target site was added into the 5 end of forward primers to improve the cloning efficiency.PCR using the following program: 4 at 95ºC, 40 cycles of 30 ' at 95ºC, 30 ' at 61ºC and 10 ' at 72ºC, and a final step of 2 at 72ºC, was performed to amplify the inserts.PCR products were purified using the MinElute PCR purification kit (Qiagen).Fragments were digested using EcoRI-HF (New England Biolabs) and purified using the MinElute Cleanup kit (Qiagen).pUAST vector was double digested using EcoRI-HF and HpaI (New England Biolabs), then the digested vector was run in an electrophoresis gel, and the band located at a 7.9 kb mark was cut and purified using the EZNA Cycle pure kit (Omega Biotek) Purified digested vector and inserts were ligated in a 1:30 vector / insert ratio using the T4 DNA ligase (New England Biolabs) following manufacturer's protocol.Then, they were transformed into DH5a competent cells (Invitrogen) and antibiotic-resistant clones were sequenced.CR40469 -and CR34335 -containing pUAST vectors were injected into the VK33 position at the FlyORF Drosophila Injection Service.

Embryo-to-adult viability and pupariation assays
Flies aged 3-5 days were allowed to lay eggs on standard medium in plates containing yeast paste for 3 h at 25ºC.The eggs were incubated for 16 h at 25ºC.Then, the unhatched embryos were transferred to vials containing standard medium in four replicates containing exactly 25 embryos each, resulting in a total of 100 embryos per condition.
For the analysis of pupariation, the vials were incubated at 25ºC and the number of pupae was counted every 12 h starting 108 h after egg laying.The pupariation percentage at each timepoint was calculated according to the total number of larvae capable of transitioning to the next developmental phase.
To assess embryo-to-adult viability, the vials were incubated at 25ºC for 15 days and the number of hatched adults per vial was counted.Embryo-to-adult viability was calculated as the proportion of adults hatched from the deposited embryos.The proportion of hatched females was calculated as the percentage of females among all the hatched adult flies.

Analyses of adult body size and wing phenotypes
Flies aged 3-5 days were allowed to lay eggs on standard medium in vials for 3 h at 25ºC.The eggs were incubated at 25ºC until adulthood.Then, the adult flies were placed at -20ºC for 5 min for immobilization and images from the lateral view were taken under a microscope.Twelve adult flies per condition were imaged.The lengths of the head, thorax and abdomen were summed up to obtain a score for the adult body size.
For the study of wing phenotypes, female flies of appropriate genotypes were selected and stored for at least 24 h in a 1:2 glycerol:ethanol solution.Then, wings were dissected in water, washed in ethanol, mounted in 6:5 lactic acid:ethanol and analyzed under a microscope.Wing size was determined as the area inside the perimeter of the wing blade.Wings were considered aberrant or non-regenerated when missing complete veins and / or when notches were present in the wing blade.

RNA extraction, reverse transcription and quantitative PCR (qPCR)
For RNA extraction, 50 wing discs per sample were dissected in Schneider's medium (Sigma Aldrich).The Quick-RNA Microprep kit (Zymo Research) was used following the manufacturer's instructions to isolate RNA, then it was incubated with DNase I (Promega) at 37ºC for 30 minutes and treated with the RNA Clean and Concentrator-5 kit (Zymo Research).A total of 1 μg of RNA was used as a template for cDNA synthesis using Moloney Murine Leukemia Virus reverse transcriptase (M-MLV RT) (Invitrogen).
Reactions containing FastStart Universal SYBR Green Master (Rox) (Roche) and the appropriate cDNA and primers were run in a 7500 Real-Time PCR System (Applied Biosystems).Samples were normalized to the levels of sply and fold changes were calculated using the ddCt method.Three technical replicates were used for each reaction, and three separate biological replicates were collected for each experiment.The designed primer sequences are shown in Supplemental Table S5 .

Induction of cell death and transgene activation
To study regeneration, we used the wing specific sal E / Pv enhancer to drive the expression of LHG in the cells of the central part of the wing disc ( sal E / Pv -LHG ), where the proapoptotic construct lexO-rpr was activated.The LHG is a modified version of lexA that is suppressible by thermosensitive Gal80 (Gal80 TS ) ( 52 ).We used an additional tub-Gal80 TS construct to inhibit the expression of rpr at 17ºC.
Flies were allowed to lay eggs on standard medium in vials for 6 h at 17ºC.Embryos were incubated at 17ºC for 192 h (8 days), before being transferred to a water bath at 29ºC for 11 h for rpr activation.Subsequently, they were returned to 17ºC until adulthood.Controls lacking the lexO-rpr transgene were treated in parallel.Additional controls with the identical genotypes were incubated at 17ºC until adulthood.Wing dissection and handling were performed as described above.
We used nub-Gal4 to induce the expression of UAS-CR40469 or UAS-CR3433 5 in the pouch of wing imaginal discs.For constitutive activation of the transgenes, embryos were cultured at 25ºC.In situ hybridization analysis was performed after 96 h (4 days) of incubation, while the analysis of adult wings was performed after 15 days.To investigate the effect of transgene activation in the context of regeneration, we followed the same protocol described above using nub-Gal4 and sal E / Pv -LHG in combination with tub-Gal80 TS to prevent the expression of both constructs at 17ºC.

RNA-seq sample collection and RNA isolation
For RNA-seq sample collection, flies were allowed to lay eggs on standard medium in vials for 6 h at 17ºC.The discs were incubated at 17ºC for 192 h (8 days), then the vials were transferred to a water bath at 29ºC for 16 h to activate rpr expression.L3 larvae from the following genotypes were selected: lexO-rpr ; sal E / Pv -LHG:tub-Gal80 TS (control) and CR40469 −/ − ; lexO-rpr ; sal E / Pv -LHG:tub-Gal80 TS ( CR40469 homozygous mutants).
Fifty wing discs per sample were dissected in cold Schneider's medium.The Quick-RNA Microprep kit (Zymo Research) was used following manufacturer's instructions to isolate the RNA.Then, it was incubated with DNase I (Promega) at 37ºC for 30 minutes and treated with the RNA Clean and Concentrator-5 kit (Zymo Research).The purity and concentration of the resulting RNA were assessed using Nanodrop (Thermo Fisher Scientific) and Qubit (Invitrogen).

RNA-seq library preparation and sequencing
For library preparation, 500 ng of total RNA were used for reverse transcription.Ribosomal RNA (rRNA) was depleted by selecting the poly-A transcripts.All libraries were sequenced on an Illumina HiSeq2500 sequencer, using 50-bp paired-end reads.Library preparation and sequencing were performed at the Genomics Unit of the Center for Genomic Regulation (CRG).
Mapping and assembling pipeline of CR40469 −/ − RNA-seq data Transcriptomic data were processed using the grape-nf pipeline ( https:// github.com/guigolab/ grape-nf).Reads were aligned to the fly genome (dm6) using STAR v2.4.0j ( 47 ), with up to 4 mismatches per paired alignment, using the FlyBase genome annotation version r6.29.Only the alignments mapping to ten or fewer loci are reported.Genes and transcripts were quantified in TPMs using RSEM v1.2.21 ( 46 ).GTF version r6.29 contains a total of 16412 genes: 13957 PCGs and 2455 lncRNAs.In our study, lncRNAs were defined as noncoding genes with > 200 bp and aligned to canonical chromosomes.
Quality control of the alignment sequencing data was performed using QualiMap v.2.2.1 ( 53 ) and Picard v.2.6.0 ( http: // broadinstitute.github.io/picard ).Using QualiMap, we obtained the number of reads, the number of mapped reads, the duplication rate and the GC percentage.We obtained the dropout and GC dropout using Picard.Assessment of the reliability of the replicates was measured with weighted correlation network analysis (WGCNA).WGCNA was implemented with the R package WGCNA v1.69 ( 54 ).A cutoff of less than 2 standard deviations from a normal distribution was implemented to use a replicate.The number of mapped reads and the correlation between replicates are provided in Supplemental Table S6 and Supplemental Figure S7 , respectively.
Filtering pipeline and differential expression analysis of CR40469 −/ − RNA-seq data We used the statistical methods implemented in DESeq2 v1.26.0 ( 42 ).Only genes with an expression of at least 1 TPM in at least one sample were selected for the differential expression analysis.The two-factor with interaction approach was implemented, considering the following design matrix: genotype, condition and genotype ∼condition, where the genotype is control or CR40469 −/ − and the condition is regeneration or non-regeneration.All genes with an absolute fold change > 1.7 and a Benjamini-Hochberg adjusted P -value < 0.05 were considered differentially expressed.

Multiple sequence alignment
The alignment of the CR40469 and CR34335 transcripts was performed using Clustal Omega ( 55 ).We used the unique CR40469 transcript and the two annotated transcripts of CR34335 as input.The alignment was run using the default parameters.

Conservation of CR40469 and CR34335 in other Drosophila species
To analyze the conservation of CR40469 and CR34335 in other Drosophila species, we used the Multiz alignments tool of the UCSC Genome Browser ( 56 ).For each block of conservation selected, we extracted the genomic sequence of each species and used Clustal Omega ( 55 ) to obtain the multiple sequence alignment.Positions identical to the Drosophila melanogaster sequence were represented in red.

Riboprobe synthesis
To analyze the localization of the CR40469 and CR34335 transcripts, we synthesized a digoxigenin (DIG)-labeled RNA probe.For this, we selected the 214-bp sequence of the CR40469 transcript, which is 99.1% identical to the CR34335 transcripts, for PCR amplification.We named this probe 214-probe .gDNA from Canton S larvae was used as a template for probe synthesis.Fragments were amplified by PCR using the primer sets reported in Supplemental Table S5 .The T3 promoter sequence and an EcoRI target site were added to the 5 end of the forward primer, while the T7 promoter sequence and a KpnI target site were added to the 5 end of the reverse primer.Amplicons were purified using the MinElute PCR Purification kit (Qiagen).Then, we digested the amplicons using EcoRI (antisense probe) or KpnI (sense probe), and added calf intestinal alkaline phosphatase (Promega) to prevent religation.DIG-labeled probes were prepared using a DIG RNA labeling mixture (Roche) and the T7 (antisense probe) or T3 RNA polymerase (sense probe).Synthesized probes were purified using the RNA Clean and Concentrator-5 kit (Zymo Research).The size of the probes was confirmed running an agarose gel.

Fluorescence in situ hybridization
The FISH protocol used in this work is a slightly modified version of the protocol described in Jandura and colleagues ( 57 ).Freshly dissected wing imaginal discs were fixed (30 min with 4% formaldehyde and 0.1% picric acid) and washed with PBTT (PBS with 0.1% Tween-20, 0.3% Triton X-100 and 0.1% picric acid).To quench endogenous peroxidase activity, the samples were incubated twice for 15 min with 0.3% hydrogen peroxidase in PBS and washed with PBTT.Then, samples were incubated with 80% acetone in PBS for 10 min at -20ºC, washed with PBTT and rinsed with 1:1 PBTT:hybridization solution (50% formamide, 5 × SSC buffer, 0.1% Tween-20, 0.3% Triton-X-100, 0.1 mg / ml of heparin and 1% salmon sperm ssRNA in DEPC water).Then, the samples were incubated with tempered pre-boiled hybridization solution for 3 h at 56ºC.The sense or antisense probe was diluted in hybridization solution (50 ng of probe per 100 μl of hybridization solution), and the mixture was denatured for 5 min at 85ºC prior to the overnight incubation at 56ºC.Then, probes were removed and the samples were washed for 15 min at 56ºC with a decreasing concentration of hybridization solution:PBTT (3:1 twice, 1:1 and 1:3), followed by washes with PBTT.The samples were incubated with a blocking solution (1% BSA in PBTT) for 20 min at room temperature prior to the incubation with anti-digoxigenin (1:2000 in blocking solution) for 2 h.The anti-digoxigenin antibody was washed with blocking solution.For signal amplification purposes, the samples were incubated with the Tyramide Signal Amplification (TSA) system (Perkin Elmer) 2 h in the dark, and then washed thoroughly with PBTT.Finally, samples were incubated for 15 min with YO-PRO-1 (Invitrogen), mounted in SlowFade Antifade (Life Technologies) and imaged under a fluorescence microscope.

Image processing and analysis
Non-fluorescence images were taken using a Leica DMLB optical microscope, while fluorescence images were taken using a Leica SPE confocal microscope.All images were processed using Fiji ( 44 ) and the Adobe Illustrator software.

Statistical analyses
Prior to any statistical analysis, the Shapiro-Wilk normality test was used to check the data distribution.According to the result, the subsequent tests were parametric or nonparametric.
For comparisons of two groups, Student's t -test or the Mann-Whitney U test were used.For comparisons of > 2 groups, one-way ANOVA or the Krustal-Wallis test followed by Dunn's test for multiple comparisons were used.
To address differences in the proportion of regenerated wings, a contingency table of regenerated and nonregenerated wings followed by the Fisher exact test was used.Bonferroni correction was applied when multiple comparisons were used.

Expression of lncRNAs in developing imaginal discs
We analyzed the expression profiles of the 2455 lncRNAs annotated in the Drosophila genome (FlyBase genome annotation version r6.29) in imaginal discs and compared them with those of the protein-coding genes (PCGs) using available transcriptomic data from wing, leg and eye discs at three different developmental stages ( 49 ): third instar larvae (L3), early pupae (EP) and late pupae (LP) (Figure 1 A).The numbers of expressed lncRNAs and PCGs were similar across all the tissues and stages: ∼200 expressed lncRNAs and ∼8000 expressed PCGs per condition, which represent ∼8% of annotated lncRNAs and 57% of annotated PCGs, respectively (Figure 1 B).In accordance with previous observations, the expression levels of lncRNAs were remarkably lower compared to those of PCGs ( Supplemental Figure S1 A-A").No differences were observed in terms of transcript length, number of exons or GC content comparing expressed and non-expressed lncR-NAs ( Supplemental Figure S1 B-E).Most expressed lncRNAs and PCGs were present in the wing, leg and eye discs at the three developmental stages studied, highlighting the genetic similarity of these tissues (Figure 1 C, D).However, we observed a higher tissue-(Figure 1 C) and stage-specificity (Figure 1 D) for lncRNAs compared to PCGs.The stage-specificity was higher at the LP stage for lncRNAs and PCGs, consistent with the morphogenetic events taking place during metamorphosis ( 28 ).To get insight into the expression profiles of lncRNAs and PCGs during imaginal disc development, we standardized the expression values of each gene across all developmental samples.For PCGs, we identified four major gene clusters: (i) genes more expressed at the LP stage, (ii) genes more expressed at the L3 and EP stages of wing and leg discs, (iii) genes more expressed in the eye, particularly at the LP stage, and (iv) genes more expressed at the L3 and EP stages of eye discs (Figure 1 E).On the contrary, lncRNAs formed two main clusters: (i) lncRNAs more expressed at LP stages, and (ii) lncRNAs more expressed at L3 and EP stages (Figure 1 F).
We next classified the expressed lncRNAs as genic (if they were in the exonic or intronic regions of PCGs) or intergenic, and paired each lncRNA with their closest PCG neighbor, independently of the direction of transcription.In this way, in-tergenic lncRNAs were paired with their closest PCG, while genic lncRNAs were associated with their overlapping PCG (Figure 2 A).Due to the high compactness of the Drosophila genome, intergenic lncRNAs were paired with PCGs located at a median distance of 2.1 kb, with 36% of pairs located less than 1 kb apart.We categorized the expression profile of each lncRNA according to its changes over time from L3 to EP and LP, classifying them into 4 groups: increasing, decreasing, peak or valley (Figure 2 B; Supplemental Figure S1 F, G).The same categorization was applied to their associated PCGs.Each lncRNA-PCG association was then classified as concordant if the classification of both genes matched, discordant if their classification was opposite (increasing-decreasing and peak-valley), and unrelated if they were not concordant or discordant or if the associated PCG was not expressed at any stage.The proportions of concordant and discordant pairs were quite similar across the tissues (Figure 2 C-C") ( Supplemental Table S1 ), and we found a significant positive correlation for lncRNA-PCG concordant pairs and a significant negative correlation for discordant pairs (Figure 2 D-D").
We performed a Gene Ontology (GO) term enrichment analysis of the concordant PCGs.In the wing disc, we observed an enrichment in genes involved in development, including segmentation and wing disc morphogenesis (Figure 2 E).For instance, the coding gene silver ( svr ), which contributes to the proper formation of the adult wing in terms of shape and size ( 58 ), showed a concordant developmental profile with the lncRNA CR44892 , which is in an antisense orientation within the first intron of svr (Figure 2 F).Concordant PCGs from the leg disc were enriched in several developmentrelated processes, such as neuroblast and muscle cell differentiation, as well as imaginal disc-derived leg morphogenesis (Figure 2 E').The PCG ftz transcription factor 1 ( ftz-f1 ), which participates in leg development in response to steroid hormone activation ( 59 ), showed a comparable expression profile to the intronic lncRNA CR45938 (Figure 2 F').In the eye disc, we found an enrichment in phototransduction-related genes, which are required for the proper development of the eye disc (Figure 2 E").One of these genes, the PCG optic ganglion reduced ( ogre ), participates in the formation of the gap junctions needed for the transduction of light signals from the photoreceptor to the central nervous system ( 60 ,61 ), and shows a concordant expression profile with the exonic antisense lncRNA CR44084 (Figure 2 F").These concordant lncRNA-PCG associations may reflect either the sharing of gene-regulatory elements, or the action in cis of the lncRNAs in the modulation of the expression of the overlapping PCGs.

Expression of lncRNAs in regenerating wing discs
Since lncRNAs seem to play important roles in cellular responses to stress, we analyzed the expression profiles of lncR-NAs in wing imaginal discs after the induction of cell death using the proapoptotic gene reaper ( rpr ).We used previously obtained RNA-seq data from different stages of damage recovery: early, mid and late stages corresponding to 0, 15 and 25 h after stopping cell death induction, respectively ( 39 ) (Figure 3 A).To obtain a robust list of differentially expressed (DE) lncRNAs, we performed pairwise comparisons between control and regeneration samples for each time-point.A highconfidence list of 131 DE lncRNAs in regeneration was obtained ( Supplemental Table S2 ).We found 29 upregulated and 33 downregulated lncRNAs in the early stage of regeneration,  followed by 11 upregulated and 56 downregulated lncRNAs in the mid stage, and 22 upregulated and 30 downregulated in the late stage (Figure 3 B).The majority of these lncRNAs were DE at one time-point (71.8%, 94 genes), while 9.9% (13 genes) were DE throughout the entire regeneration process (Figure 3 B).Around half of DE lncRNAs were intergenic (48.8%), while genic lncRNAs were located predominantly in exonic regions (29.8%) compared to introns (21.4%) (Figure 3 C).No significant differences were observed between DE and non-DE lncRNAs in terms of intergenic / genic ratio, transcript length or number of exons ( Supplemental Figure S2 A-D).
Next, we classified the 131 DE lncRNAs according to their expression profile across time in regeneration (Figure 3 D), and associated each of them with their overlapping or closest PCG.This resulted in 131 lncRNA-PCG pairs (131 unique lncRNAs and 121 unique PCGs), 20.6% of which were concordant, 14.5% were discordant, and 64.9% were unrelated (Figure 3 E; Supplemental Table S3 ).As expected, we observed a high positive correlation for lncRNA-PCG concordant pairs and a strong negative correlation for discordant pairs compared to unrelated pairs (Figure 3 F).We highlighted the concordant pair composed by the natural antisense transcript CR44811 , which is known to regulate the isoform usage of the overlapping blistered ( bs ) gene ( 11 ), and the discordant intergenic lncRNA CR44677 and CG9300, which showed a high negative correlation in comparison with the unrelated pair composed by CR9284 and tpr (Figure 3 G).
To better characterize the lncRNAs DE in regeneration, we analyzed their expression during normal development in imaginal discs ( 49 ).Our analysis identified 4 distinct clusters of lncRNAs: those exhibiting peak expression in the wing disc at the L3 stage (cluster 1), those with decreasing expression at the LP stage (cluster 2), those with increasing expression at the LP stage (cluster 3), and those with increased expression in the eye (cluster 4) (Figure 3 H,I).Approximately two-thirds (64%) of lncRNAs DE in regeneration belong to early development clusters 1 and 2, while 19% and 17% are part of clusters 3 and 4, respectively.We did not observe differences in the cluster distribution between upregulated and downregulated genes.The dynamic expression profiles throughout development indicate a tight regulation and a potential developmental role of DE lncRNAs.However, when we expanded our analysis to the context of regeneration, we noticed that these developmental clusters were not maintained ( Supplemental Figure S2 E, F), indicating a context-dependent regulation of their expression.

LncRNA CR40469 is not required for normal development but plays a role during wing regeneration
From the set of 131 DE lncRNAs, we searched for putative candidate genes suitable for generating a mutant.To minimize any interference on overlapping genes, we only considered intergenic lncRNAs.We focused on lncRNAs upregulated during early regeneration, particularly the lncRNA CR40469 (Figure 4 A).CR40469 is a monoexonic lncRNA spanning 214 bp and localized in the subtelomeric region of the X chromosome, positioned roughly 120 kb away from the chromosome end.It is generally not expressed throughout development, yet it can be detected in the L3 wing disc ( 49 ,62 ).Based on the criteria from Figure 3 E, CR40469 was classified as unrelated, as the closest PCG, CG17636 , is not expressed in regeneration.
We generated a knockout mutant by using the ends-out homologous recombination technique, deleting the entire CR40469 locus as well as 663 bp upstream and 212 bp downstream (deleting a total region of 1089 bp), and replacing them with an mCherry cassette ( Supplemental Figure S3 A, B).Homozygous mutant animals were fully viable (Figure 4 B), reached adulthood at the same sex proportions as the wild type flies (Figure 4 C), and did not show differences in adult body size compared to wild types (Figure 4 D), indicating that the deletion of CR40469 did not compromise the normal development of male and female flies.We did not find differences in the pupariation time either (Figure 4 E), indicating that mutants did not undergo developmental delays.Also, the deletion of CR40469 did not affect the adult wing size or morphology (Figure 4 F, G).Thus, we concluded that CR40469 is not required for fly development under normal laboratory conditions.
Since we identified CR40469 as a lncRNA upregulated in wing regeneration, we tested the effects of knocking out CR40469 after tissue damage.For this, we genetically activated the proapoptotic gene rpr in the central zone of the wing disc ( spalt domain).We used the LHG / LexO system, which allows conditional inactivation by the temperature sensitive Gal80 TS (Figure 5 A).We did not observe defects in the wing patterning when flies were maintained at permissive temperature (17ºC) ( Supplemental Figure S4 A).However, after cell death induction, mutant flies showed a significant reduction in their wing regeneration capacity, as there was a higher proportion of aberrant non-regenerated wings characterized by major disruptions in the vein patterning, such as the absence of complete sets of veins and crossveins as well as the appearance of notches in the margins of the wing blade (Figure 5 B, C).
To characterize the molecular changes occurring in CR40469 mutants after cell death induction in the wing discs, we analyzed by RNA-seq the gene expression profiles at the early stage of regeneration.Non-mutant regenerating discs were used as controls.We found a total of 95 DE genes after cell death induction in the CR40469 mutants: 48 of which were upregulated and 47 downregulated (Figure 5 D; Supplemental Table S4 ).While it remains unclear if certain of these genes are essential for wing regeneration, some of them were previously found upregulated in response to injury or stress, such as NijA ( 63 ) or Cyp6a17 ( 64 ), while others are predicted to play a role in response to ROS or infection, including Karl , Lgr4 , Nep1 and NT1 .The aberrant expression of some of these genes may lead to defective regeneration.
To determine whether the deletion of CR40469 had a local effect on gene expression, we mapped the genomic position of the DE genes along the X chromosome.The 112 genes located up to 908 kb downstream of the deleted region showed no significant changes in gene expression, with the closest upregulated and downregulated genes located 1.23 Mb and 923 kb away from the CR40469 locus, respectively (Figure 5 E).We next conducted qPCR analysis to examine the expression of genes located within a 100 kb vicinity of the deletion site.Our results confirmed those obtained from the RNA-seq analysis.Specifically, none of the genes analyzed showed altered expression in regenerating CR40469 mutant wings compared to the wild type flies (Figure 5 F).Thus, we concluded that the absence of CR40469 had no significant impact on the expression of nearest genes during regeneration.

CR40469 is duplicated in the Drosophila melanogaster genome
To further characterize CR40469 , we performed a BLAST search to assess its putative conservation, but we could not identify a similar sequence in any other species, including other Drosophila species.However, we identified the entire CR40469 genomic sequence with 99.1% similarity (212 / 214 identities) within the annotated lncRNA CR34335 sequence in the Drosophila melanogaster genome (Figure 6 A; Supplemental Figure S3 C).CR34335 is located 3.2 Mb downstream of the CR40469 locus in the X chromosome and is positioned inside a long intron of the PCG Dpr-interacting protein a ( DIP-a) , which is not expressed in development or regeneration in the wing disc ( 49 ).The CR34335 gene is monoexonic and contains two polyadenylation signals (Figure 6 A), giving rise to two predicted transcripts that span 249 and 361 nucleotides.Both isoforms contain the entire sequence identical to CR40469 .Despite their sequence similarity, CR40469 and CR34335 exhibit opposite expression patterns.In the development of wing, leg, and eye discs, CR40469 shows minimal expression, while CR34335 is highly expressed (Figure 6 B).Conversely, during wing disc regeneration, CR40469 is upregulated at early and late stages, whereas CR34335 is consistently downregulated across all time points (Figure 6 C).Using the Multiz alignment tool ( 56 ), we explored the conservation of both lncRNAs in other Drosophila species.No traces of synteny nor sequence homology were found for the CR40469 locus, however, small regions of high similarity were detected for CR34335 (Figure 6 D).Particularly, we highlighted two conserved blocks located at the edges of the CR34335 sequence shared with CR40469 (Figure 6 D).The high conservation of these regions in evolutionarily close Drosophila species, the absence of conservation of the CR40469 locus, and the high sequence identity of CR34335 and CR40469 in Drosophila melanogaster , suggest that the CR40469 lncRNA might have originated from the CR34335 locus specifically in the Drosophila melanogaster species.We next analyzed an available CR34335 mutant that had an insertion of a 7-kb transposon within its exonic sequence, affecting both isoforms.To understand the impact of the transposon insertion, we examined by qPCR the expression of CR34335 and the 7 genes located up to 100 kb upstream and downstream of the insertion site.We confirmed the absence of CR34335 expression in the CR34335 mutant and noted the downregulation of 4 nearby genes (Figure 6 E).Homozygous mutant flies were fully viable and, despite a slight reduction in size ( ∼3%) compared to wild type wings (Figure 6 F,G), we did not detect any abnormality in the shape or vein patterning (Figure 6 F).
To test whether CR34335 is necessary for wing regeneration, we used the same system as described above to transiently activate rpr in the spalt domain of the wing disc.At permissive temperature, mutants developed normal wings ( Supplemental Figure S4 A).Upon rpr activation, we did not observe a significant decrease in the regeneration capacity of CR34335 mutants compared to controls (Figure 6 H,I), indicating that CR34335 is dispensable for regeneration.
Finally, since subcellular localization is one of the primary factors determining lncRNA function, we performed fluorescence in situ hybridization (FISH) using a probe complementary to both lncRNAs ( 214-probe ).A strong signal was found in the cytoplasm of wild type discs, and it was even stronger in the CR40469 mutants, indicating that CR34335 is a cytoplasmic lncRNA ( Supplemental Figure S5 A-D).Nevertheless, probably due to its lower expression levels in development, we could not detect CR40469 transcripts in the wing imaginal disc ( Supplemental Figure S5 E, F).

Regeneration mutant phenotypes are rescued by ectopic expression of CR40469
To elucidate whether the ectopic expression of CR40469 or CR34335 was sufficient to restore the regeneration capacity of CR40469 homozygous mutants, we generated transgenic flies carrying a copy of the CR40469 or the CR34335 genes downstream of a UAS sequence.To assess the efficiency of the transgenes, we performed in situ hybridization using a nubbin driver ( nub-Gal4 ) to induce their expression in the pouch of the wing disc in a CR34335 homozygous mutant background.This choice was made to avoid the potential masking effect of high CR34335 levels on the detection of the ectopic expression of the transgenes.Similar to the endogenous CR34335 ( Supplemental Figure S5 A-D), we observed ectopic CR34335 also in the cytoplasm (Figure 7 A).Also, upon inducing CR40469 expression in CR34335 mutants, a cytoplasmic signal was detected (Figure 7 A), suggesting that the endogenous CR40469 might also be located in the cytoplasm.We next analyzed the effects of the ectopic expression of CR40469 and CR34335 in the wing in a wild type background using nub-Gal4 , whose domain encompasses the entire wing blade in the adult.In both cases, adult wings showed normal vein patterning (Figure 7 B) and size (Figure 7 C), indicating that the overexpression of CR40469 or CR34335 does not affect normal wing development.Similarly, no visible defects were detected in adult flies when using a ubiquitous driver (data not shown).Next, we tested whether expression of CR40469 or CR34335 could rescue the aberrant wing phenotypes observed upon the activation of cell death in CR40469 homozygous mutants.We combined the induction of cell death in the spalt domain with the expression of CR40469 or CR34335 in the nub domain.The ectopic expression of CR40469 significantly increased the proportion of regenerated wings observed in CR40469 mutant flies following the induction of cell death (Figure 7 D, E).Additionally, CR40469 expression led to significantly larger wings compared to controls, indicating that not only vein patterning but also wing size is recovered (Figure 7 F).On the other hand, expressing CR34335 in the nub domain of CR40469 mutants also resulted in a higher number of regenerated wings, although wing size was not significantly restored (Figure 7 D-F).Taken together, our findings point to a potential trans -acting role of CR40469 during the damage response, with CR34335 potentially performing a partially redundant function in regeneration when CR40469 is absent.

Discussion
Non-coding transcripts are being increasingly identified in transcriptional studies and there is growing evidence of lncRNA functionality.It is likely that the most expressed lncRNAs are actively or potentially involved in several cellular processes ( 7 ).Here, with the aim of describing the participation of lncRNAs in development and regeneration, we have studied their expression in Drosophila imaginal discs.Compared to the around 8000 PCGs expressed in the discs, we have only detected the expression of around 200 lncRNAs.As previously reported for Drosophila and other species ( 1 , 3 , 8 ), the tissue and stage specificity of lncR-NAs is remarkably higher than that of PCGs regardless of the tissue or developmental stage analyzed, implying a tight control of lncRNA transcription in the developing imaginal discs.
One of the most studied functions of lncRNAs is ability to influence gene expression at multiple levels, including the modulation of chromatin accessibility, the activation of RNA polymerase II, the recruitment of transcription factors, and the regulation of mRNA elongation ( 9 ).LncRNAs act in trans , functioning far and independently of their transcription site ( 65 ,66 ), or in cis , acting near and depending on their locus ( 67 ).In humans, the expression of lncRNAs tends to correlate with the expression of overlapping and genomically close PCGs ( 1 ).Currently, there is no similar data in Drosophila , but the higher genome compactness may reinforce the same idea.Here, we have associated each lncRNA expressed in the developing discs with their overlapping or closest PCG.Regardless of the tissue type, ∼25% of the analyzed pairs have shown concordant expression profiles in development.We postulate that some of these concordant lncR-NAs could regulate the expression of their associated PCG in cis .Consequently, we have analyzed the functions of the concordant PCGs to infer the biological processes in which their paired lncRNAs might participate.Our findings highlight that lncRNAs expressed in the developing discs are preferentially located near PCGs involved in key developmental processes, such as wing disc morphogenesis in the wing disc, phototransduction in the eye disc, and imaginal disc-derived leg morphogenesis in the leg disc.Although we cannot discard the fact that some of the concordant lncRNA-PCG pairs might just reflect the sharing of the same regulatory elements, some of these lncRNAs could be involved in the regulation of key PCGs during development.
Evaluating the functionality of lncRNAs presents significant challenges, and the effects of their lack of function can only be determined conclusively after thorough monitoring of the potential phenotypic impact across various conditions.We chose to study lncRNAs expression and function following damage because of the dynamic changes that take place during the process of regeneration.A few studies have undertaken global analyses of lncRNAs in regeneration, for instance following injury in skeletal muscle ( 68 ) or during cardiac regeneration in zebrafish ( 69 ).Specific lncRNAs have also been linked to regeneration: H19 is associated with nerve degeneration and regeneration in rats ( 70 ), and mucosal regeneration in mice ( 71 ); lncMREF is a positive regulator of muscle regeneration in mice, pigs and humans ( 72 ); CR46040 has recently been identified as crucial for the proliferation of intestinal stem cells in response to injury in Drosophila ( 73 ).In the context of wing regeneration, we have identified a subset of 131 DE lncRNAs that could participate in the recovery process.In line with their high specificity, most lncRNAs are DE in one particular time-point, suggesting that they might be required only during specific periods.In contrast to the peak of active transcription described for PCGs ( 39 ), DE lncRNAs tend to be downregulated in regeneration rather than upregulated, suggesting that coding and non-coding genes are regulated differently in response to damage.
We have focused our study on the intergenic lncRNA CR40469, which is upregulated after damage in the wing imaginal disc and is required for the regeneration process.CR40469 is exclusively found in Drosophila melanogaster and its sequence is included within the sequence of the lncRNA CR34335 , whose 5 and 3 ends are conserved in a variety of Drosophila species.This led us to hypothesize that CR40469 may have emerged from the CR34335 locus through a duplication event specific to Drosophila melanogaster .In fact, CR40469 is located in the subtelomeric region of the X chromosome, immediately after the most proximal HeT-A element, one of the retrotransposons responsible for telomere elongation in flies ( 74 ,75 ), reinforcing the hypothetical scenario of retrotransposition involving CR34335 , which is located 3.2 Mb downstream.Both genes show an opposite expression pattern: while CR40469 is probably silenced within heterochromatin in most cells and conditions, CR34335 is highly and ubiquitously expressed at similar levels of genes encoding for ribosomal subunits ( Supplemental Figure S6 A, B).Upon cell death, however, CR40469 is upregulated and CR34335 is downregulated.This contrasting expression profile, coupled with their high sequence similarity, suggests that these lncRNAs might engage in competitive binding for specific factors involved in regulating their expression or stability.
The subcellular localization of lncRNAs is crucial in determining their molecular function ( 76 ).We have detected CR34335 transcripts in the cytoplasm.Although we could not detect endogenous CR40469 transcripts, we have identified ectopic CR40469 also in the cytoplasm, indicating that both lncRNAs are exported from the nucleus.Multiple studies have indicated that the majority of cytoplasmic lncRNAs localize to ribosomes ( 77 ,78 ).It is speculated that some of these lncRNAs may be translated into small peptides ( 79 ), some of which have been shown to be functional (80)(81)(82).Indeed, a short peptide consisting of 33 amino acids is predicted from both CR40469 and CR34335 ( Supplemental Figure S6 C).The likelihood that this small peptide is actually translated is low considering that the predicted amino acid sequence is included in the region shared by CR40469 and CR34335, which is not conserved in other species ( Supplemental Figure S6 C) and no peptide domains can be inferred.Moreover, its amino acid sequence does not match peptides identified in proteomics experiments ( 83 ), which is unusual given the high expression levels of CR34335 .Thus, although CR34335 was identified in the polysomal fraction of early embryos ( 84 ) and S2 cells ( 83 ), this association is not evidence of translation, as some lncRNAs are known to regulate mRNA translation and stability by binding to ribosomes ( 76 ), which could be relevant to the roles of CR40469 and CR34335 .It is also important to consider the influence of the extra nucleotides at the 5 and 3 ends of CR34335 compared to CR40469 , which might result in diverse secondary structures or binding associations, thus potentially affecting their molecular function.
In addition to its subcellular localization, a trans -acting role for CR40469 in regeneration may be implied by the seemingly stochastic distribution of differentially expressed genes in CR40469 mutants across the Drosophila genome.The restoration of the regeneration capacity in CR40469 mutants following ectopic expression of CR40469 further supports its trans -acting role.Moreover, the observation that ectopic expression of CR34335 is able to partially rescue the regeneration capacity of CR40469 mutants suggests a potential functional substitution of CR34335 in the absence of CR40469 .In conclusion, our study identifies CR40469 as a trans -acting cytoplasmic lncRNA participating in wing regeneration.Additionally, as the non-coding genome remains largely unexplored, the partial duplication of lncRNAs that we have uncovered in our study may not necessarily be an exceptional phenomenon, but an instance of a more general mechanism by means of which lncRNAs acquire novel functions.
D), leg disc (Figure 2 D'), eye disc (Figure 2 D") or regeneration samples (Figure 3 F) was used for the PCC calculation.

Figure 1 .
Figure 1.Expressed lncRNAs and PCGs in Drosophila imaginal discs.( A ) Representation of the developmental samples used for the gene expression analysis.( B ) Number of protein-coding genes (PCGs) and lncRNAs expressed in the wing, leg and eye discs in third instar larvae (L3), early pupae (EP) and late pupae (LP).( C ) Tissue specificity of PCGs and lncRNAs in L3 (left), EP (middle) and LP (right).( D ) Stage specificity of PCGs and lncRNAs in the wing (left), leg (middle) and e y e discs (right).( E, F ) Expression of (E) PCGs or (F) lncRNAs in de v elopment.Gene expression is normalized to Z -score values.Genes not expressed in any sample were not considered.Complete hierarchical clustering was used to identify the gene clusters.

Figure 2 .Figure 3 .
Figure 2. Association of lncRNAs with PCGs in the de v eloping wing, leg and e y e discs.( A ) Association of genic and intergenic lncRNAs with their o v erlapping and closest PCGs, respectively.( B ) Classification of the expressed lncRNAs in the wing disc according to their developmental expression profile.( C ) Classification of all the lncRNA-PCG pairs in the wing (C), leg (C') and e y e (C") discs.( D ) Pairwise Pearson correlation coefficient of concordant, discordant and unrelated lncRNA-PCG pairs in the wing (D), leg (D') and e y e (D") discs.( E ) Gene Ontology (GO) term enrichment analysis of the PCGs from the concordant lncRNA-PCG pairs in the wing (E), leg (E') and e y e (E") discs.( F ) Examples of concordant lncRNA-PCG pairs from the wing disc morphogenesis term (F), imaginal disc-derived leg morphogenesis term (F'), and phototransduction term (F").The genomic positioning of the lncRNA and its associated PCG is represented.(***) P < 0.001.

Figure 4 .
Figure 4. Deletion of CR40469 does not affect fly de v elopment.( A ) Expression of the 13 intergenic and early upregulated lncRNAs in early control and early regeneration.( B ) Embryo-to-adult viability presented as the proportion of hatched adults from deposited embryos.Four replicates of N = 25 each were used.( C ) Proportion of hatched females from the emerging adults.Four replicates of N = 25 each were used.( D ) Adult body size analysis by measuring the length from the head to the abdomen.N = 12 per condition.( E ) Pupariation analysis showing the proportion of flies that reached the pupal stage at each time-point.Four replicates of N = 25 each were used.( F ) Sample images of the adult wings of wild type flies and CR40469 mutants incubated at 25ºC.Scale bar = 250 μm. ( G ) Wing size of the wings represented in F measured as the wing blade area.The mean and standard error of the mean (SEM) are presented in B, C and E. The mean and standard deviation (SD) are presented in D and G. n.s.= non-significant.

Figure 5 .
Figure 5. Wing regeneration is impaired in the CR40469 mutants.( A ) Schematic representation of the method used to analyze the regeneration capacity of flies.Cell death was induced in the spalt domain of the wing disc for 11 h at the L3 stage, then flies w ere allo w ed to reco v er until adulthood.RNA-seq of control and CR40469 homozygous mutants were performed following cell death induction.( B ) Sample images of most common phenotypes of regenerated and aberrant wings in control and CR40469 mutants.The number of wings per phenotype is represented.Scale bar = 250 μm. ( C ) Percentage of regenerated wings from B. ( D ) Volcano plot displaying the upregulated (red), downregulated (blue) and non-DE genes (gray) in regenerating mutants compared to regenerating controls.( E ) Distances of all genes located on the X chromosome relative to the position of CR40469 .Upregulated, downregulated and non-DE genes are shown in red, blue and gray, respectively.( F ) Expression analysis by qPCR of genes located < 100 kb from the CR40469 locus in the wild type and homozygous mutant wing discs.3 biological replicates are measured.The mean and SD are represented in C and F.

Figure 6 .
Figure 6.The sequence of CR40469 is duplicated in the Drosophila genome.( A ) Schematic representation showing the genomic position of the lncRNAs CR40469 and CR34335 .( B-C ) Expression profile of CR40469 and CR34335 in de v elopment and regeneration.Normalized expression is represented as log 10 (TPMs + 0.1).Pairwise Pearson correlation coefficient of both lncRNAs is shown.( D ) Multiz alignments of the CR34335 locus in different Drosophila species.The genomic sequence of blocks 1 and 2 are represented, as well as their sequence identity compared with the Drosophila melanogaster sequence.( E ) Expression of CR34335 and the genes located < 100 kb a w a y in the control and CR34335 mutant wing discs measured by qPCR. 3 biological replicates per condition are measured.( F ) Sample images of the adult wings of wild type flies and CR34335 mutants incubated at 25ºC. ( G ) Wing size of the wings represented in F measured as the wing blade area.( H ) Sample images of most common phenotypes of regenerated and aberrant wings in control and CR34335 mutants.The number of wings per phenotype is represented.( I ) Percentage of regenerated wings from H. The mean and SD are represented in E, G and I. Scale bars in F and H = 250 μm.(***) P < 0.001; (*) P < 0.05; n.s.= non-significant.

Figure 7 .
Figure 7. Ectopic expression of CR40469 rescues the CR40469 mutant regeneration phenotypes.( A ) In situ hybridization of the wing imaginal disc in control (top row) or after expression of CR40469 (middle row) or CR34335 (bottom row) driven by a nub promoter in a CR34335 homozygous mutant bac kground.21 4-probe is complementary to both CR40469 and CR34335 .N ≥ 4 per condition.Scale bar = 20 μm. ( B ) Sample wings of nub > + (control), nub > CR40469 and nub > CR34335 incubated at 25ºC. ( C ) Wing size of genotypes represented in B. ( D ) Sample images of most common phenotypes of regenerated and aberrant wings of nub > + , nub > CR40469 and nub > CR34335 in a CR40469 homozygous mutant background.The number of wings per phenotype is represented.( E ) Percentage of regenerated wings from D. ( F ) Wing size of genotypes represented in D. Quartiles, interquartile range and median are represented.Regenerated and non-regenerated wings are shown in green and red, respectively.The mean and SD are represented in C and E. Scale bars in B and D = 250 μm.(***) P < 0.001; (*) P < 0.05; n.s.= non-significant.